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CRISPR/Cas9 vectors are among several types of emerging genome editing tools that can quickly and efficiently create mutations at target sites of a genome (the other two popular ones being ZFN and TALEN).
Cas9 is a member of a class of RNA-guided DNA nucleases which are part of a natural prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophage. Within the cell, the Cas9 enzyme forms a complex with a guide RNA (gRNA), which provides targeting specificity through direct interaction with homologous 18-22nt target sequences in the genome. Hybridization of the gRNA to the target site localizes Cas9, which then cuts the target site in the genome.
To achieve CRISPR-mediated gene targeting it is essential for the target cells to co-express both Cas9 as well as the target site-specific gRNA at the same time. This can be accomplished by either expressing both Cas9 and the gRNA sequence from the same vector (a.k.a. all-in-one vector) or by using separate vectors for driving Cas9 and gRNA expression (Cas9 only and gRNA only vectors respectively). The advantage of using separate vectors over an all-in-one vector for expressing Cas9 and gRNA is that it offers the flexibility of combinatorial usage of different gRNA expression vectors in conjunction with a variety of Cas9 variants (wild type nuclease, nickase, nuclease-dead) depending upon the user’s experimental goal.
We offer multiple variants of the most widely used SpCas9 derived from Streptococcus pyogenes in our Cas9 nuclease collection to facilitate your vector design on our online portal. These variants include - hCas9, the humanized version of wild type SpCas9 which efficiently generates double-strand breaks (DSBs) at target sites; hCas9-D10A, the “nickase” mutant form of hCas9 which generates only single-stranded cuts in DNA; dCas9, a catalytically inactive variant of SpCas9, bearing both D10A and H840A mutations; SpCas9-HF1, a high-fidelity variant of SpCas9; and eSpCas9, an enhanced specificity variant of SpCas9. Fusions of dCas9 with activation domains such as dCas9/VP64 and dCas9/VPR or with repression domains such as dCas9/KRAB are also available for CRISPRa and CRISPRi applications, respectively. Additionally, we offer SaCas9 derived from Staphylococcus aureus for applications requiring a shorter Cas9 variant compared to Spcas9 and AsCpf1 derived from Acidaminococcus for achieving DNA cleavage via staggered DNA double stand breaks.
The AAV Cas9 expression vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with helper plasmids, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. The Cas9 expression cassette placed in-between the two ITRs is introduced into target cells along with the rest of viral genome. A user-selected promoter drives Cas9 expression, which is then directed to the DNA target site of interest in the presence of the target site-specific gRNA sequence.
The wild-type AAV genome is a linear single-stranded DNA (ssDNA) with two ITRs forming a hairpin structure on each end. It is therefore also known as ssAAV. In order to express genes on ssAAV vectors in host cells, the ssDNA genome needs to first be converted to double-stranded DNA (dsDNA) through two pathways: 1) synthesis of second-strand DNA by the DNA polymerase machinery of host cells using the existing ssDNA genome as the template and the 3' ITR as the priming site; 2) formation of intermolecular dsDNA between the plus- and minus-strand ssAAV genomes. The former pathway is the dominant one.
AAV genomic DNA forms episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers can remain for the life of the host cells. In dividing cells, AAV DNA is lost through the dilution effect of cell division, because the episomal DNA does not replicate alongside host cell DNA. Random integration of AAV DNA into the host genome can occur but is extremely rare. This is desirable in many gene therapy settings where the potential oncogenic effect of vector integration can pose a significant concern.
A major practical advantage of AAV is that in most cases AAV can be handled in biosafety level 1 (BSL1) facilities. This is due to AAV being inherently replication-deficient, producing little or no inflammation, and causing no known human disease. Due to their low immunogenicity in host organisms, our AAV Cas9 expression vectors are the perfect tools for in vivo CRISPR-based applications.
Many strains of AAV have been identified in nature. They are divided into different serotypes based on different antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). When our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. The serotypes currently offered by us for our AAV vector systems include - serotypes 1, 2, 3, 4, 5, 6, 6.2, 7, 8, 9, rh10, DJ, DJ/8, PHP.eB, PHP.S, AAV2-retro and AAV2-QuadYF. The table below lists different AAV serotypes and their tissue tropism.
|AAV1||Smooth muscle, skeletal muscle, CNS, brain, lung, retina, inner ear, pancreas, heart, liver|
|AAV2||Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes|
|AAV3||Smooth muscle, liver, lung|
|AAV4||CNS, retina, lung, kidney, heart|
|AAV5||Smooth muscle, CNS, brain, lung, retina, heart|
|AAV6||Smooth muscle, heart, lung, pancreas, adipose, liver|
|AAV6.2||Lung, liver, inner ear|
|AAV7||Smooth muscle, retina, CNS, brain, liver|
|AAV8||Smooth muscle, CNS, brain, retina, inner ear, liver, pancreas, heart, kidney, adipose|
|AAV9||Smooth muscle, skeletal muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney, adipose|
|AAVrh10||Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney|
|AAV-DJ||Liver, heart, kidney, spleen|
|AAV-DJ/8||Liver, brain, spleen, kidney|
|AAV2-QuadYF||Endothelial cell, retina|
|AAV2.7m8||Retina, inner ear|
|Tissue type||Recommended AAV serotypes|
|Smooth muscle||AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10|
|Skeletal muscle||AAV1, AAV9|
|CNS||AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB|
|Brain||AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8|
|Retina||AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2-QuadYF, AAV2.7m8|
|Inner ear||AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8|
|Lung||AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10|
|Liver||AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8|
|Pancreas||AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10|
|Heart||AAV1, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ|
|Kidney||AAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8|
|Adipose||AAV6, AAV8, AAV9|
For further information about this vector system, please refer to the papers below.
|Science. 339:819 (2013)||Description of genome editing using the CRISPR/Cas9 system|
|Nat. Biotech. 31:827 (2013)||Specificity of RNA-guided Cas9 nucleases|
|Nat. Commun. 9:1911 (2018)||Review on various Cas9 variants|
|Science. 351:400 (2016)||AAV-mediated CRISPR/Cas9 editing|
Our AAV Cas9 expression vector is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression. This viral vector can be packaged into virus using all known capsid serotypes, is capable of very high transduction efficiency, and presents low safety risk.
Flexibility: The AAV Cas9 only vector can be co-transduced with multiple different gRNA sequences for targeting different genomic sites of interest.
Safety: AAV is the safest viral vector system available. AAV is inherently replication-deficient and is not known to cause any human diseases.
Low risk of host genome disruption: Upon transduction into host cells, AAV vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.
High viral titer: Our AAV vectors can be packaged into high titer virus. When AAV virus is obtained through our virus packaging service, titer can reach >1013 genome copy per ml (GC/ml).
Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our AAV vector when it is packaged into the appropriate serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).
Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.
Difficulty transducing certain cell types: Our AAV vector system can transduce many different cell types including non-dividing cells when packaged into the appropriate serotype. However, different AAV serotypes have tropism for different cell types, and certain cell types may be hard to be transduced by any serotype.
Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.
PAM requirement: CRISPR/Cas9 based targeting is dependent on a strict requirement for a protospacer adjacent motif (PAM), located on the immediate 3’ end of the gRNA recognition sequence. The required PAM sequence varies depending on the Cas9 variant being used.
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
Promoter: The promoter that drives the expression of the downstream Cas9 gene is placed here.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of the Cas9 nuclease variant chosen by the user.
Regulatory element: Allows the user to add the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). WPRE enhances virus stability in packaging cells, leading to higher titer of packaged virus and enhances expression of transgenes.
BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.